Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes.
Proteasomes are one of the cellular complexes controlling protein degradation from archaebacteria to mammalian cells. We recently purified and characterized the catalytic core of the proteasome, the 20S form, from Trypanosoma brucei, a flagellated protozoa which causes African trypanosomiasis. To identify the role of proteasomes in African trypanosomes, we used lactacystin, a specific inhibitor of proteasome activity. Lactacystin showed potent inhibition of the activity of 20S proteasomes purified from both bloodstream and procyclic (insect) forms of T. brucei (IC50 = 1 microM). It also inhibited proliferation of T. brucei cells in culture assays, with 1 microM inhibiting growth of bloodstream forms, whereas 5 microM was required to block proliferation of procyclic forms. Analysis of the DNA content of these cells by flow cytometry showed that 5 microM lactacystin arrested procyclic cells in the G2 + M phases of the cell cycle. Fluorescence microscopy revealed that most of the cells had one nucleus and one kinetoplast each, indicating that the cells had replicated their DNA, but failed to undergo mitosis. This suggests that transition from G2 to M phase was blocked. On the other hand, incubation of bloodstream forms with 1 microM lactacystin led to arrest of 30-35% of the cell population in G1 and 55-60% of the cells in G2, indicating that both transition from G1 to S and from G2 to M were blocked. These observations were also confirmed by using another inhibitor of proteasome, N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV), which arrested procyclic forms in G2, and bloodstream forms in both G1 and G2. These results suggest that proteasome activity is essential for driving cell cycle progression in T. brucei, and that proteasomes may control cellular functions differently in bloodstream and procyclic forms of T. brucei.
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Department of Pharmaceutical Chemistry, University of California San Francisco, 94143-0446, USA. mutomba@cgl.ucsf.edu
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D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000032:analysis / Q000096:biosynthesis; D005434:Flow Cytometry; D007399:Interphase / Q000187:drug effects*; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008938:Mitosis / Q000187:drug effects; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D016196:S Phase / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development
D015853:Cysteine Proteinase Inhibitors; D016054:DNA, Protozoan; D007976:Leupeptins; D009097:Multienzyme Complexes; C089486:carbobenzoxy-leucyl-leucyl-norvalinal; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine